1 Adelina Buburuzan
The Effect of Substrate Concentration on Potato
Peroxidase Lab
Adelina Buburuzan
Ms. Phung SBI4U1-02
Introduction:
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2 Adelina Buburuzan
Enzymes are vital to the lives of animals and plants alike. They are a type of
proteins that speed up chemical reactions inside living organisms. They are very
important in the catalysis of certain compounds as well as establishing a rate of reaction
efficient enough to keep the organism alive. All reactions require a specific activation
energy (EA) to run the reaction to completion, and what an enzyme does is help the
reactants reach this. Substrates are the reactants in this case, and they bind to the
active sites of an enzyme either through the “lock-and-key method” or the more flexible
method of the “induced fit model”. After a product is formed, the enzyme has the useful
characteristic of being recyclable. One enzyme, peroxidase, is found in plants and its
function is to catalyze hydrogen peroxide (a harmful compound) into harmless water
and oxygen. Without this biological process triggered by peroxidase, the potato plant,
for instance, could not survive. Like all other enzymes, there are certain conditions
under which peroxidase (found inside potatoes as well as humans) can have the most
effect on its substrate. In this lab, the concentration of its substrate is observed. The
question to be answered is: how does changing the concentration of hydrogen peroxide
affect the activity of peroxidase? Evidently, the rate of reaction for the catalysis of
hydrogen peroxide is what must be determined to be able to understand if the enzyme
works better with lower or higher concentrations of substrate.
Hypothesis:
If a substrate concentration was to be increased then this change would result in an
overall faster rate of reaction. This statement can be supported by the fact that the
purpose of an enzyme is to speed up a chemical reaction. It does so by allowing its
substrate to bind to its active site. In this lab, the substrate observed is hydrogen
peroxide, and it is seen that with the peroxidase enzyme (found inside potatoes) acting
as its catalyst, it decomposes into oxygen and water. Yet, if the concentration of the
substrate were to be weak, then less substrate would be able to bind to the available
active sites all at once. Thus, the reaction would be more slow. On the other hand, if the
concentration of hydrogen peroxide is high, then more of it can bind to the enzyme-
increasing the rate of reaction. Therefore, by the addition of a stronger concentration of
hydrogen peroxide to an unchanged volume of peroxidase, the enzyme would have an
increased activity so reaction will speed up and become more efficient.
Variables:
Independent Variable- Concentration of hydrogen peroxide (since it is a changed/
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3 Adelina Buburuzan
manipulated factor)
Dependent Variable- Rate of Reaction; height of bubbles divided by time of reaction
(since it depends on the independent variable)
Controlled Variables-
1. The mass of potatoes, as they were 3g for each treatment.
2. The time, as each trial was 12 minutes in length.
3. The volume of each hydrogen peroxide solution, as it was 2 mL per treatment.
4. The measuring of the height of the bubbles, as it was from the where the solution
ended to the very last trace of bubbles.
5. The pH of the solution stays the same throughout.
6. The temperature is room temperature and stays constant.
Materials:
● Cut-up potatoes (3g)
● 1 test tube rack
● 6% and 30% hydrogen peroxide
● Water
● Safety goggles
● Digital scale
● Weight boat
● Min. 4 test tubes per trial
● Droppers
● 1 Scoopula
● 200 mL beaker
● 15 cm ruler
● Labelling tape
● 1 Timer
Procedure:
1. Safety goggles were put on.
2. Using a test tube rack, 4 test tubes were collected, and labelled with: 6%, 12%,
18% and 24%.
3. Using the scoopula, potatoes were placed on the weight boat.
4. The weight boat was placed on the digital scale which was used to measure 3g
of potato for each test tube.
5. Using a scoopula, the potato mass was placed into each test tube.
6. An additional 4 test tubes were obtained to prepare to pour the substrate into the
test tubes with the potatoes.
7. 3 mL of 6% hydrogen peroxide was added to an empty test tube with a dropper,
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4 Adelina Buburuzan
and labelled with tape.
8. A new test tube was prepared with the same mass of potato.
9. Inside a 200 mL beaker, a 12% concentrated hydrogen peroxide was created
using water and the 30% solution that was available.
10. 3 mL of 12% diluted hydrogen peroxide was added to another test tube, and
labelled with tape.
11. Steps 7-8 were repeated for a 18%/ 3 mL sample and a 24%/ 3 mL sample.
12. All four different hydrogen peroxide concentrations were poured into the test
tubes with the potatoes at the same exact time.
13. Timer was started, and the solutions were left to react for 12 minutes.
14. The height of the bubbles formed was measured with at the 12 minute mark with
a ruler- measuring from where the bubbles start on top of the solution up to the
last trace of them.
15. Procedure was repeated for two more trials.
16. All results were recorded. Hydrogen Peroxide was disposed of in the waste bin,
and the potatoes in the garbage.
17. The rest of the materials were put back in their places, and workplace was
cleaned up.
Observation Tables:
Height of Bubbles Measured During Each Trial
Hydrogen Peroxide TRIAL ONE (height TRIAL TWO (height TRIAL THREE
Concentrations in cm) in cm) (height in cm)
6% 0.75 2.9 2.6
12% 3.0 3.4 3.7
18% 2.9 3.1 4.5
24% 2.5 3.0 3.5
Qualitative Observations Made Throughout Experiment
Colour/ Texture Temperature Other
-All final solutions (after the -The higher the -The 12% and 18% H202
12 mins were finished) had concentration, the warmer were close in height of
a milky, translucent, light- the test tube felt after the bubbles at the halfway-
mark (6%). By the end,
white physical appearance chemical reaction.
they were the highest of all
were the bubbles were. the treatments.
-Bubbles were foamy. -In a separate trial with the
-Potatoes were shades of 24% alone, the bubbles
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5 Adelina Buburuzan
light orange/brown. were still weak/ short in
-Yellow potato starch height, showing no
stayed at the bottom of the discrepancy from trials 1-3.
test tube.
Results:
Sample Calculation
For the 6% H202:
Average of 2.1cm of bubbles/ 12 minutes= 0.175cm/ min.
Substrate Concentration Experiment Results
Concentration of Hydrogen Average Height of Bubbles Rate of Reaction (cm/min)
Peroxide Substrate(%) (cm)
6% 2.1 0.175
12% 3.4 0.283
18% 3.5 0.292
24% 3.0 0.250
Discussions/ Analysis:
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6 Adelina Buburuzan
There are many interesting points to be observed from the graph above
concerning the concentration of a substrate in relation to the rate of reaction of its
interaction with its enzyme. Firstly, there is an initial fairly steep positive slope going
from the 6% concentration of substrate to the 12%. This indicates that the rate of
reaction increases after the concentration becomes 6% stronger. Continuing across the
graph, the slope is positive, substantially less steep and nearly approaches zero/ nearly
reaching a plateau interval on the line graph. Finally, from 18% to 24% hydrogen
peroxide concentration, the slope for rate of reaction changes to negative, but is also
steeper than the last interval. However, the first interval’s slope is still steeper than the
other ones. Looking at each concentration value individually, the 6% hydrogen peroxide
had the slowest rate of reaction (in cm/min), followed by the 12% and 18%. After
peaking at the 18%, the 24% treatment was shown to have a slower rate of reaction
than all the others except for the 6% substrate.
One can interpret many things from analyzing the behaviour of the graph. For
instance, it is certain that with a substrate of weak concentration, the enzyme present
will work on all the substrate that it can to catalyze it into water and oxygen. However,
there is not much substrate present in the 6% solution. Therefore, the rate of reaction is
much slower compared to a more strongly concentrated substrate such as the 12% or
18%. In these two solutions, the ratio of substrate to enzyme is higher so more
substrates are binding to the active sites of more enzymes, causing the chemical
reaction to be quicker and more efficient. As for the 24% being lower than the previous
two treatments, this is due to the enzyme activity plateau-ing before beginning to
decrease/ slow down. Somewhere in the midst of this interval- between 18% to 24%
concentration- there was likely one specific concentration that caused the slope to
plateau before going in the negative direction. Peroxidase has a unique concentration
that gives it its maximum rate of reaction, and this is what is called its maximum velocity.
What occurs during a plateau of this type is that the enzyme in each potato becomes
saturated with hydrogen peroxide, meaning it has no space for any more to bind to it.
Thus, the catalysis of such high concentrations of the hydrogen peroxide is no longer
occurring at an efficient rate as there is excess of the substrate floating around while all
the active sites are already occupied. Unfortunately, the graph showing a negative slope
on the far right side is not entirely common, but has previously been found in other
studies. A possible explanation for the behaviour of the graph after the point of plateau
is that after the enzyme becomes fully saturated, the excess hydrogen peroxide
interacts with the active sites so much that it actually reduces them in size.
Consequently, this would cause the curve to have a downwards/ negative slope.
Overall, there was a lot to be learned from the results of this experiment.
Conclusion:
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7 Adelina Buburuzan
After the completion and analysis of this lab, it is clear that the hypothesis was
correct. The stronger the concentration of hydrogen peroxide, the greater the activity of
peroxidase and thus the greater the rate of reaction. This chemical reaction of catalysis
was sped up as the substrate concentration was increased due to interacting more with
the enzyme that was meant to catalyze it. The enzyme peroxidase was able to fulfill its
function as a protein within the potatoes, and break the hydrogen peroxide down into
harmless water and oxygen at a faster rate when the concentration was increased.
However, an amendment should be made to the original hypothesis as it was
discovered that for every enzyme, there is a point where its rate of reaction plateaus.
This occurs after the concentration reaches a specific point. In this case, the point was
found somewhere between the 18% and 24% concentration of hydrogen peroxide. Its
maximum velocity was not clearly seen, but is known that it has one as all enzymes do.
It is at this point where an enzyme’s activation energy is brought to the lowest possible
point, making the rate of reaction the most efficient possible. It is thus an interesting
piece of information that the lower concentrations of hydrogen peroxide, a harmful
substance, are actually catalyzed more slowly by peroxidase inside living organisms.
It must be stated that a perfect plateau was not seen in the graph due to the human
limitation of not having treatments of closer and numerically more substrate
concentrations. Another limitation that was present was that the measuring tools did not
have the highest degree of accuracy as the beakers in which the hydrogen peroxide
solutions were diluted had no markers at all. There were simply none available at the
time of dilution. This affected the results, decreasing their accuracy substantially, as the
volume measurements for the different concentrations of substrate were not exact. For
the future, having and using a more accurate measuring device such as a 10 or 20 mL
graduated cylinder would be recommended. Yet, the biological limitations of this lab
must be taken into account as well. The first to be discussed is that the volume of
potatoes could not be kept constant as they were all cut in different sizes. Bigger
potatoes contained more peroxidase, and vice versa. This certainly affected the amount
of peroxidase found in each treatment, which was an important factor in the catalysis of
the hydrogen peroxides of different concentrations and thus the overall rate of reaction
for each of the trials.This tarnished the final results of the experiment by creating
inconsistencies within the trials. To have a more accurate lab, the potatoes would all
need to be cut in the same size so each treatment could be identical. An alternative
would be to have many more trials in order for the average of all of them to have a more
accurate reading despite not knowing exactly how much peroxidase was in each
treatment.
Finally, during the dilution, another limitation was present which was that tap water was
used for each treatment. Evidently, this posed the risk of separate chemical reactions
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8 Adelina Buburuzan
occurring during the experiment due to all the ions found in tap water. These ions may
have very well also acted as cofactors- chemicals such as calcium and zinc (in their
inorganic ion form) that may become nonprotein components of an enzyme and
increase its activity. Again, this would hurt the results obtained. A fairly easy solution to
this dilemma is to used filtered water. To conclude, despite the limitations, it was an
informative lab on the effects of substrate concentration on enzyme activity that
revealed that revealed that the faster, the higher the rate of reaction of hydrogen
peroxide getting catalyzed by peroxidase. In general, it is seen how enzymes are
sensitive to external factors, especially to their substrates, which they closely interact
with. As mentioned earlier, they are incredibly important proteins. Therefore, it is not
surprising that they require special conditions to function at their maximum potential.
Bibliography:
1. Http://ljournal.ru/wp-content/uploads/2017/03/a-2017-023.pdf. (2017).
doi:10.18411/a-2017-023
2. Basics of enzyme kinetics graphs. (n.d.). Retrieved February 24, 2018, from
https://www.khanacademy.org/science/biology/energy-and-enzymes/enzyme-
regulation/a/basics-of-enzyme-kinetics-graphs
3. Basics of enzyme kinetics graphs. (n.d.). Retrieved February 24, 2018, from
https://www.khanacademy.org/science/biology/energy-and-enzymes/enzyme-
regulation/a/basics-of-enzyme-kinetics-graphs
4. Peroxidase. (n.d.). Retrieved February 24, 2018, from
https://www.sciencedirect.com/topics/neuroscience/peroxidase
5. (n.d.). Retrieved February 26, 2018, from
https://www.nku.edu/~whitsonma/Bio150LSite/Lab
%2011%20Enzymes/Bio150LEnzymes.html
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